Therapeutic agents for renal diseases and method for screening the same

ABSTRACT

The present invention provides a method for screening or identifying therapeutic or prophylactic agents for renal diseases, which comprises assaying a test substance for the activity of up-regulating the expression of fatty acid-binding protein (FABP), and novel mouse proximal renal tubular epithelial cell lines useful therein. The present invention also provides therapeutic or prophylactic agents for renal diseases comprising, as an active ingredient, an agent having activity of up-regulating FABP expression; agents for up-regulating the expression of FABP, and for treating or preventing renal diseases, which comprise a compound having activity of peroxisome proliferator-activated receptor (PPAR) agonist or carnitine palmitoyltransferase (CPT) inhibitor or the like.

TECHNICAL FIELD

[0001] The present invention relates to therapeutic agents for renaldiseases and a method for screening and identifying the same. Inparticular, the present invention relates to a method for screening andidentifying such agents, which method was established with closeattention directed to the expression of fatty acid-binding protein. Thepresent invention also relates to a cell line established from kidney(proximal renal tubule) cells.

BACKGROUND ART

[0002] Renal diseases such as nephritis generally presents complex anddifferent pathological aspects, and, when become chronic, may causeserious progression including glomerulosclerosis or interstitialfibrosis, and eventually renal insufficiency. Accordingly, anappropriate treatment in the earlier stage is highly demanded, but thereare few therapeutic drugs effective for such purpose. Among the limitednumber of available drugs, steroids show clear effect but can beaccompanied by unacceptably strong adverse side effects. Accordingly,there have been great demands for novel and superior therapeutic agentsfor renal diseases.

[0003] Fatty acid-binding proteins (FABPs) are classified as a group ofproteins of molecular weight of around 15 kDa, which present in cytosoland have ability to bind to fatty acids. FABP has been thought to play arole in the regulation of metabolic enzymatic system by transporting andaccumulating fatty acids into cells. However, nothing has been knownabout the correlation between FABP and renal diseases so far.

[0004] As for FABP, at least seven molecular species are known, such asliver-type FABP (L-FABP), intestinal-type FABP (1-FABP), heartmuscle-type FABP (H-FABP), brain-type FABP (B-FABP),cutaneous/epidermal-type FABP (C-FABP/E-FABP), adipocyte-type FABP (aP2)and peripheral neuron-type FABP (myelin P2). They all have bindingactivity to fatty acids and considered to be members of a family evolvedfrom a common ancestral gene. Each FABP shows specific histologicaldistribution pattern. The nomenclature of respective FABP means in whichorgan the FABP was firstly found, but does not necessarily mean that thesaid FABP exclusively exists in such organ.

[0005] In human kidney, at least two FABPs, i.e., liver-type (L-FABP)and heart muscle-type (H-FABP), are expressed and L-FABP is distributedmainly in proximal renal tubule while H-FABP in distal renal tubule(Maatman et al., Biochemical Journal, vol. 288, p. 285-290, 1992;Maatman et al., Biochemical Journal, vol. 273, p. 756-766, 1991).

[0006] The expression and distribution of FABP in kidney of rodents isquite different from that of human. In rodents, L-FABP is expressedscarcely in proximal renal tubule and is distributed in distal renaltubule only in a small amount (Maatman et al., Biochemical Journal, vol.288, p. 285-290, 1992). Predominant FABPs in rodent kidney are H-FABPand kidney-type FABP (K-FABP). H-FABP is mainly distributed in distalrenal tubule. K-FABP is thought to be produced as follows. Whenα_(2u)-globulin synthesized in liver is excreted from the circulatoryblood to urine in kidney, a portion thereof is reabsorbed by renaltubular cells and converted into K-FABP through the intracellularprocessing (Kimura et al., FEBS Letters, vol. 246, p. 101-104, 1989).

DISCLOSURE OF INVENTION

[0007] One of the purposes of the present invention is to providetherapeutic or prophylactic agents for renal disease, which exert effectthrough a novel mechanism of action, and a method for screening oridentifying such drugs. The other purpose of the present invention is toprovide a novel cell line useful in the above-mentioned screening oridentifying method and the like.

[0008] As described above, there had been nothing known about thecorrelation between FABP and renal diseases before the present inventorsfound that the decrease in FABP level in urine or renal tissues precedesthe infiltration of macrophage or interstitial fibrosis in mouse modelof nephritis. The inventors have also found that the L-FABP level inrenal tissue is decreased in patients suffering from renal diseases ofbad prognosis, and established a method of diagnosing renal diseasesbased on these findings, which method is focused on FABP(JP-A-11-242026, WO99/27363). The present inventors have continued theresearch and found that the increase of expression of FABP leads totreatment of renal diseases such as nephritis, in other words, a drugcapable of up-regulating the expression of FABP can be a therapeuticagent for renal diseases, and established the present invention.

[0009] The present invention provides a method for screening oridentifying therapeutic or prophylactic agents for renal diseases, whichcomprises assaying a test substance for the activity of up-regulatingthe expression of fatty acid-binding protein (FABP) in animal cells.Further, the present invention provides therapeutic or prophylacticagents selected or identified by such method. Also provided aretherapeutic or prophylactic agents for renal diseases comprising, as anactive ingredient, an agent having activity of up-regulating theexpression of FABP in kidney cells or tissues, for example, a compoundhaving activity of peroxisome proliferator-activated receptor (PPAR)agonist or the like. The present invention also provides a novel mouseproximal renal tubular epithelial cell line useful in the said screeningor identifying method.

[0010] In patients of renal disease, tissues or cells of kidney areexposed to various kinds of stress such as high proteinuria, ischemia,etc. The therapeutic or prophylactic agents selected or identified bythe present method, namely a drug capable of up-regulating theexpression of FABP, can protect such cells or tissues. Accordingly,their mechanism of action is based on the protective activity of FABP inkidney tissue or cells (particularly, in renal tubular cells) againststress caused by high proteinuria, ischemia or the like.

[0011] Recently, the high proteinuria is regarded as not only a mereindication of renal injury but also one of risky factors per se(Kees-Folts et al., Kidney International, vol. 45, p. 1697-1709, 1994;Eddy et al., Journal of American Society of Nephrology, vol. 5, p.1273-1287, 1994). It has been known that albumin, a main component ofurine, is generally associated with several free fatty acid molecules.Fatty acids binding to urinary proteins are considered to be reabsorbedfrom brush border membrane of proximal renal tubular epithelial cells,and intracellularly bound to FABP, transported to mitochondria orperoxisome and undergone β-oxidation. In the absence of sufficientamount of FABP, normal β-oxidization of fatty acids can be hindered,which may stimulates the generation of lipid factors (kidney injuryfactor) that activate macrophages, and lead to the development ofinterstitial fibrosis via immunological mechanism. In such a case,enhancement of expression of FABP in proximal renal tubular epithelialcells can normalize the fatty acid metabolism and suppress thegeneration of lipid factor having renal injuring activity and therebycontributing to the improvement of pathology of renal disease.

BRIEF DESCRIPTION OF DRAWINGS

[0012]FIG. 1 is an electron micrograph showing the morphology of mouseproximal renal tubular epithelial cell line No.13 (37-13).

[0013]FIG. 2 shows the results of measurement of capability of mouseproximal renal tubular epithelial cell lines (No.10, 13, 14, 15, 16 and19) in responding to hormonal stimulation (ability to induceintracellular cAMP production upon stimulation with PTH and AVP).

[0014]FIG. 3 shows the results of measurement of capability of mouseproximal renal tubular epithelial cell lines (No. 13 and 16) in uptakeof BSA.

[0015]FIG. 4 shows the results of measurement of capability of testsubstances in up-regulating the expression of L-FABP in reporter assayusing mouse proximal renal tubular cell lines.

[0016]FIG. 5 shows the effect of suppression of L-FABP expression on thecytotoxicity in a model for hypoxia/re-oxygenation (in vitro renalischemia/reperfusion model). In FIG. 5, L-FABP antisense refers toLLC-PK1 cells originated from porcine proximal renal tubular cellcomprising transiently transfected L-FABP antisense RNA expressionvector, and control refers to the same cells comprising transientlytransfected vector only.

[0017]FIG. 6 shows the effect of imposed L-FABP expression on thecytotoxicity in model for hypoxia/reoxygenation (in vitro kidneyischemia/reperfusion model). In FIG. 6, “24” refers to a mouse proximalrenal tubular cell line (clone 24) (control cell), and “24-19” refers toa mouse proximal renal tubular cell line in which human L-FABP gene hasbeen introduced and forced to express (clone 24-19).

[0018]FIG. 7 shows the results of examination into the effect of a drug(MCC-555) on the proteinuria and NAG excretion in a model mouse of renaldisease (adriamycin-induced glomerulosclerosis model mouse).

[0019]FIG. 8 shows the results of examination into the effect of a drug(MCC-555) on the blood biochemical parameters (blood cholesterol andalbumin level) in a model mouse of renal disease (adriamycin-inducedglomerulosclerosis model mouse).

[0020]FIG. 9 shows the results of examination into the effect of a drug(MCC-555) on the proteinuria and NAG excretion in a model mouse forrenal disease (adriamycin-induced glomerulosclerosis model mouse)produced using a transgenic mouse introduced with human L-FABP gene.

BEST MODE FOR CARRYING OUT THE INVENTION

[0021] Renal diseases contemplated by the present invention (method ofscreening or identifying therapeutic agents, and the resultant drugs)includes renal disease such as diabetic nephropathy, glomerulonephritis,nephrotic syndrome, focal glomerulosclerosis, immune complex nephropathy(e.g., IgA nephropathy, membranous nephropathy), lupus nephritis,drug-induced renal injury, renal insufficiency, and the like.

[0022] The present invention is applicable to mammals including apes,dogs, cats, pigs, cows, sheep, goats, rabbits, rats, mice as well ashumans.

[0023] In mammals including human but for rodents, L-FABP and H-FABP areexpressed in kidney cells and tissues. L-FABP is distributed in proximalrenal tubule and H-FABP distributed mainly in distal renal tubule. Tocreate novel therapeutics for renal diseases, it is important to focusattention to FABP in kidney cells or tissues, especially, to FABP inproximal renal tubular cells, for example, L-FABP in proximal renaltubular cells in the case of human.

[0024] The method of screening or identifying therapeutic agents forrenal diseases of the present invention can be carried out in thefollowing manner. A test substance can be assayed for the up-regulatingactivity on intracellular FABP expression (hereinafter, it may bereferred to as “FABP up-regulating activity”) by culturing animal cells(mammalian cells or tissues) in the presence of the said test substanceand comparing the amount of FABP expressed in cells with that expressedin cells cultured in the absence of a test substance. Alternatively,such an assay can be conducted by administering a test substance toanimals and comparing the amount of FABP expressed in renal tissues orcells of the animal with that expressed in renal tissues or cells ofnon-treated animals.

[0025] When an increased amount of FABP is expressed in the presence ofa test substance (or in administration group), it is determined thatsaid substance has up-regulating activity corresponding to the degree ofeffect, and can be useful as therapeutics for renal diseases. Thescreening of therapeutic agents for renal diseases can be convenientlyconducted by selecting substances having high FABP up-regulatingactivity as candidates.

[0026] Further, since a candidate substance is assayed for the FABPup-regulating activity and then identified as a therapeutic agent havingFABP up-regulating activity, the mechanism of action as a therapeuticfor renal disease should be clear. As a result, a therapeutic agentproperly characterized can be provided.

[0027] Examples of method of detecting FABP expression include a methodwhere the expression of gene is measured by reporter assay using areporter protein as an indicator. Further, the expression of gene can bedetected at mRNA level. In addition, the expression of FABP can bedirectly detected in cell extract at protein level.

[0028] When conducting the measurement of gene expression by reporterassay, for example, a DNA construct comprising a FABP genetranscriptional regulatory region and a reporter gene ligated downstreamtherefrom is constructed and introduced into an appropriate animalcells. The cells are then cultured in the presence or absence of a testsubstance and the activity of reporter protein or the like in cellextract can be measured, and thereafter, the expression amounts bedetermined and compared.

[0029] The amino acid sequence of FABP and nucleotide sequence encodingthe same have been reported for various species (Veerkamp and Maatman,Prog. Lipid Res., vol. 34, pp. 17-52, 1995). For example, the sequenceinformation of L-FABP is known in regard to human cDNA (Lowe et al.,Journal of Biological Chemistry, vol. 260, pp. 3413-3417, 1985;Genbank/EMBL accession No. M10050), rat chromosomal gene (Sweetser etal., Journal of Biological Chemistry, vol. 261, pp.5553-5561, 1986;Genbank/EMBL accession No. M13501), and the like.

[0030] Accordingly, FABP cDNA or chromosomal gene can be obtained froman appropriate DNA libraries by any one of or combinations of PCRmethod, colony hybridization method, plaque hybridization method and thelike using a primer or probe designed on the basis of sequenceinformation as mentioned above.

[0031] The reporter construct and reporter plasmid containing the samecan be prepared by a conventional gene recombinant technology with atranscriptional regulatory region present in 5′ upstream region of FABPchromosomal gene and an appropriate reporter gene.

[0032] As a reporter gene, it is preferred to use a gene of an enzymewhich is stable and facilitates the quantitative determination ofactivity, although there are no limitations. Examples of such reportergenes include β-galactosidase gene (lacZ), bacterial transposon-derivedchloramphenicol acetyl transferase gene (CAT), firefly-derivedluciferase gene (Luc) and the like.

[0033] When the gene expression is detected at mRNA level, it can beconducted by, for example, extracting RNA (mRNA) from cells or tissues,and detecting and determining the mRNA transcribed from FABP gene by PCRmethod (Polymerase chain reaction method) (“PCR Protocols”, Innis M A,Gelfad D H, Sninsky J J and White T J eds., Academic Press, Sandiego,1990), RNase protection assay method (Nucleic Acid Research, vol. 12,pp. 7035-7056, 1984), Northern blotting analysis, or the like.

[0034] When the gene expression is detected at protein level, it can beconducted by, for example, immunochemical method (e.g., ELISA,immunohistochemical staining method) using anti-FABP antibody. Theantibody can be prepared by a conventional method using purified FABP asan antigen. The distribution among organs, molecular weight, and primarystructure of respective molecular species of FABP have been reported(Fujii et al., Arteriosclerosis, vol. 24, pp. 353-361, 1996; Veerkampand Maatman, vol. 34, pp. 17-52, 1995; Drickamer et al., J. Biol. Chem.,vol. 256, pp. 3634-3636, 1981; Unterman et al., Proc. Natl. Acad. Sci.USA, vol. 78, pp. 3478-3482). Purified FABP can be prepared on the basisof such information. Alternatively, the preparation may be conducted bygene recombinant technology using cDNA isolated on the basis of theknown nucleotide sequence of a gene.

[0035] When the present method is carried out in vitro using culturedcells, it is preferred to use animal cells (mammal cells or the like).Above all, cells from kidney of animals are preferred, renal tubularcells are more preferred and proximal renal tubular epithelial cells areespecially preferred. Cells to be used may be primary cultured cells orimmortalized cells (cell lines). Immortalized cells (cell lines) areadvantageously used in view of facilitated cultivation and handling.

[0036] As human kidney-derived cells, urinary exfoliated cells(specifically, proximal renal tubular epithelial cells) isolated fromhuman urine sample can be used. Cells from human kidney areadvantageously used to make prediction of action in human more reliable.

[0037] Specific examples of known cell lines from renal tubule includeMDCK (ATCC CRL 6253), a cell line derived from canine renal tubule(distal renal tubule), LLC-PK1 (ATCC CRL 1392), a cell line derived fromporcine renal tubule (proximal renal tubule), and the like.

[0038] In addition to the above, there are immortalized cell lines whichthe present inventors have established from mouse proximal renal tubularcells as hereinafter described in Example 2, such as mouse cell linemProx 37-13 that was originally deposited at The National Institute ofBioscience and Human Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan(Accession No. FERM P-16985; date of deposition: Sep. 9, 1998) andtransferred to an international deposition (Accession No. FERM BP-7038;date of transfer to international deposition: Feb. 21, 2000) under theprovision of Budapest Treaty. These cell lines are characterized by thefollowing properties (1), (2) and (3):

[0039] (1) it shows morphology of epithelial cells;

[0040] (2) it has ability to uptake albumin; and

[0041] (3) it is responsive to the stimulation by parathormone andinduced to produce intracellular cAMP but is unresponsive to thestimulation by vasopressin.

[0042] Among the above-mentioned renal tubule-derived cell lines,LLC-PK1 does not maintain the property (3) that is characteristic toproximal renal tubular cells in living body, although said cell line isepithelial cells originated from proximal renal tubule. See, Example 2below. In contrast to this, the cell lines that have been established bythe present inventors definitely maintain the entire propertiescharacteristic to proximal renal tubular epithelial cells includingresponding activity to hormone, and hence the use thereof should makethe system more close to the in vivo physiological environment.Accordingly, these cells can be advantageously used to establish aneffective method of screening and identifying therapeutic agents forrenal diseases.

[0043] Test substances identified to have the FABP up-regulatingactivity according to the method of the present invention can besubjected to further examination using any of known pathological models(in vivo or in vitro) to confirm the therapeutic and/or preventiveeffect.

[0044] Examples of in vitro pathological model include a modelgenerating kidney injuring lipid (inflammatory lipid) (Kees-Folts etal., Kidney International, vol. 45, pp. 1697-1709, 1994) and the like.Examples of in vivo pathological model include models of acceleratedanti-GBM nephritis (Nagai, et al., Jpn. J. Pharmacol., vol. 32, pp.1117-1124, 1982), STZ-induced diabetic nephropathy (Sharma et al.,Diabetes, vol. 45, pp. 522-530, 1996), puromycin-induced focalglomerulosclerosis (Hirano, et al., Nephron, vol. 60, pp. 443-447,1992), adriamycin-induced glomerulosclerosis (Chen, et al., Nephron,vol. 78, pp. 440-452, 1998), cyclosporin nephropathy (Gillum, et al.,Transplant, vol. 46, pp. 285-292, 1988) and the like.

[0045] Examples of drugs found to have FABP up-regulating activityinclude agonists of peroxisome proliferator-activated receptor (PPAR),carnitine palmitoyltransferase (CPT) inhibitors, and the like. Theexistence of a peroxisome proliferator-responding sequence upstream fromthe FABP gene supports the finding that PPAR agonist has up-regulatingactivity on FABP expression.

[0046] Examples of PPAR agonist usable include known PPAR agonists(Lehmann, et al., Journal of Biological Chemistry, vol. 270, pp.12953-12956, 1995; Willson et al., Journal of Medicinal Chemistry, vol.39, pp. 665-668, 1996), and also compounds which are newly confirmed tohave activity as PPAR agonist according to a method described inliteratures (WO99/10532; WO96/33724; WO96/22884; Mizukami et al.,Biochemical Biophysical Research Communications, vol. 240, pp. 61-64,1997; Krey et al., Molecular Endocrinology, vol. 11, pp. 779-791, 1997;Buckle et al., Bioorganic & Medicinal Chemistry Letters, vol. 6, pp.2121-2126. 1996; Tontonoz et al., Genes and Development, vol.8, pp.1224-1234, 1994). Above all, MCC-555 (chemical name:5-[6-(2-fluorobenzyloxy)naphthalene-2-ylmethyl]thiazolidine-2,4-dione;EP604983) is especially preferred as PPAR (PPARγ) agonist.

[0047] Examples of CPT inhibitor usable include known CPT inhibitors(Current Pharmaceutical Design, vol. 4, pp. 1-15, 1998), and alsocompounds which are newly confirmed to have CPT inhibitory activityaccording to a method described in literatures (Saeed, et al., Arch.Biochem. Biophys., vol. 305, pp. 307-312, 1993; Kanamura et al., LifeScience, vol., 37, pp. 217-223, 1985; Shinagawa et al., J. Med. Chem.,vol. 30, pp. 1458-1463, 1987). Above all, Etomoxir (chemical name: ethyl2-[6-(4-chlorophenoxy)hexyl]oxiranecarboxylate; EP46590 and CurrentPharmaceutical Design, vol. 4, pp. 1-15, 1998) and 4-THA (chemical name:2-hydroxy-3-propyl-4-[6-(tetrazol-5-yl)hexyloxy]acetophenone; Biochem.J., vol. 252, pp. 409-414, 1988) are especially preferred as a CPT (CPTI) inhibitor.

[0048] PPAR agonists and CPT inhibitors are known as therapeutic drugsfor diabetes. Further, there have been reported that symptoms of renaldiseases due to diabetes can be ameliorated with these drugs inpathological models of diabetes (Buckingham et al., Diabetes, vol. 47,pp. 1326-1334, 1998). However, this can be attributed to theamelioration of diabetes which is the primary disease and nothing hasbeen taught or suggested that PPAR agonists or CPT inhibitors areapplicable to renal diseases not caused by diabetes. To the contrary,the therapeutic or prophylactic agents of the present invention aresuitable for application to renal diseases other than those caused bydiabetes (e.g., diabetic nephropathy).

[0049] When a test substance, an agent having FABP up-regulatingactivity, a PPAR agonist, a CPT inhibitor or the like is administered toan animal or human, any administration method such as oral, intravenous,intramuscular or subcutaneous administration is available. In addition,it may be formulated together with an appropriate inert carrierdepending on the administration form, if necessary. Although anappropriate dosage varies depending on the administration route, age,body weight, conditions of the patient and the like, the daily dosagecan generally be determined within range of 1-300 mg/kg for oraladministration and 0.01-50 mg/kg for non-oral administration.

[0050] The pharmaceutical agents (therapeutics or prophylactics) of thepresent invention exert their in vivo medicinal action on the basis ofthe FABP up-regulating activity, i.e., action as PPAR agonist or CPTinhibitor. The pharmaceutical agents of the present invention do notinclude any drugs lacking sufficient FABP up-regulating activity (oraction as PPAR agonist or CPT inhibitor) to render in vivo medicinaleffect. Also excluded from the pharmaceutical agents of the presentinvention are those which exert in vivo medicinal action based on agiven main action different from FABP up-regulating activity (action asPPAR agonist or CPT inhibitor). As used herein, the pharmaceuticalagents of the present invention do not include those which areinapplicable to treatment or prophylaxis of diseases due to toxicity orinferior natures from the phrmacokinetic viewpoint or the like.

[0051] The following Examples are provided to further illustrate thepresent invention in detail but are not to be construed as limiting thescope thereof.

[0052] Throughout the following examples, otherwise mentioned, eachprocedure was conducted following the teachings in “Molecular Cloning,Sambrook, J., Fritsch, E. F. and Maniatis, T. eds., Cold Spring HarborLaboratory Press, 1989), and the reagents or kits of commercial sourcewere used in accordance with the manufactures protocol.

EXAMPLES Example 1 Preparation of L-FABP Reporter Plasmid

[0053] (1) Isolation of Human FABP cDNA

[0054] Human L-FABP cDNA was isolated from human hepatocyte cDNA library(Clontech, Cat#HL1115b LOT#5621) by PCR (polymerase chain reaction)method. Primers were designed based on known sequence information (Loweet. al., Journal of Biological Chemistry, vol. 260, pp. 3413-3417, 1985;Genbank/EMBL accession No. M10050) to result in fragment obtained by PCRhaving BamHI recognition sites at both ends. DNA fragment (about 420base pairs) resulted from PCR contained the entire human L-FABP cDNAcoding region. This fragment was ligated into an appropriate plasmid.

[0055] (2) Isolation of Human FABP Chromosomal Gene

[0056] Human L-FABP chromosomal gene was cloned from human chromosomalDNA library (Clontech, Cat#HL1006d LOT#5621) by plaque hybridizationmethod using the fragment (about 420 base pairs, BamHI fragment)obtained in (1) above which includes human FABP cDNA as a probe. Theresultant clone contained an about 20 kb insert fragment. Partialsequencing showed that this fragment included the entire human L-FABPcoding region.

[0057] A fragment (about 5360 bp, SalI-kpnI fragment) including the 5′upstream region was excised from the insert fragment, inserted into avector plasmid and used in the following experimentation. The nucleotidesequence of the fragment (SalI-kpnl fragment) including the 5′ upstreamregion is shown in the SEQ ID NO: 1.

[0058] (3) Preparation of L-FABP Reporter Plasmid

[0059] A fragment (about 100 bp) was obtained by amplifying a regionextending from the NspV recognition site (nucleotide No. 4670 in the SEQID NO: 1, in the 5′ upstream sequence) to the site positioned justbefore the initiation codon, which is about 100 bp downstream from theformer by PCR method using L-FABP gene fragment (about 5360 base pairs,SalI-KpnI fragment) obtained in (2) as a template. Separately, theSalI-KpnI fragment was introduced into the SalI-KpnI site of pUC18plasmid, and then the resulted plasmid was digested with NspV and KpnIto delete a portion downstream from the NspV recognition site. Thisplasmid was ligated to the previously obtained PCR fragment to give aplasmid pUC18-9/25.

[0060] The resulted plasmid was then digested with SalI and KpnI, andthe generated fragment (about 4760 bp) was inserted into vector plasmidpGV-B (TOYO INK) to give a plasmid for reporter assay. The insertfragment contained the sequence corresponding to the portion fromnucleotide No. 1 to No.4760 (just before the initiation codon) in SEQ IDNO: 1 with KpnI recognition sequence (6 bp) added at the 3′ terminal.

[0061] The resulted L-FABP reporter plasmid has the reporterconstruction which comprises 5′ upstream region includingtranscriptional regulatory region of human L-FABP gene and a luciferasegene (except for promoter region) ligated at the downstream therefrom.

Example 2 Primary Culture and Immortalization of Renal Tubular Cells,and Introduction of L-FABP Reporter Construct into the Same

[0062] (1) Isolation of Nephron

[0063] A nephron was isolated from mouse kidney by microdissectionmethod in the following manner. A mouse was anesthetized withpentobarbital before laparotomy and perfused with 20 ml of cold Hank'sbuffered solution (HBS) containing 0.1% BSA via abdominal artery. Then,10 ml of cold HBS containing 0.1% BSA and 0.1% collagenase type 1 wasperfused. These perfusion solutions were previously saturated with a gasmixture of 5% CO₂-95% O₂. Kidneys were then isolated and cut into slicesections of 0.5-1.0 mm thick with Surgical Brade. These sections weresoaked into 10 ml of 0.1% collagenase in HBS, incubated at 37° C. for 10minutes for enzymatic treatment and then washed with HBS twice. A singlesegment of nephrons was isolated from these slice sections whileobserving with stereomicroscope under ice cooling.

[0064] (2) Primary Culture of Renal Tubular Epithelial Cells

[0065] Primary culture of renal tubular cells was performed using thesingle segment of nephrons obtained in item (1) above in the followingmanner. The single segment was washed twice with K1 medium (50:50DMEM/Ham's F-12, 15 mM Hepes, 13.4 mM sodium bicarbonate, 5 μg/mlinsulin, 5 μg/ml transferrin, 5 ng/ml selenous acid, 0.05 μMhydrocortisone, 10 ng/ml epidermal growth factor) containing 10% fetalcalf serum, seeded into 96-well plate and cultured overnight in the samemedium. On the next day, feeder layer (2.5-5×10³ cells/well) were addedand co-cultured with the single segment. The used feeder layer was mouserenal mesenchyme-derived cell (cell line prepared by isolating cellsfrom mouse kidney with density gradient centrifugation method andsubject to immortalization) or mouse NIH 3T3 cell lacking for growthcapacity as a result of X-radiation. The used medium was 10% fetal calfserum-containing K1 medium supplied with an equivalent amount ofsupernatant of cultured feeder layer and HGF (Hepatocyte Growth Factor)(25 ng/ml). Plates used were those pre-coated with 0.1% gelatin. After4- to 6-day-cultivation, outgrowth of epithelial cells (renal tubularepithelial cells) from the segment was observed under microscope.

[0066] (3) Immortalization of Renal Tubular Epithelial Cells

[0067] Renal tubular epithelial cells were cultured in the same manneras (2) above.

[0068] After confirmation of outgrowth of epithelial cells, the cellswere transfected with a plasmid which contains SV40 Large T antigen geneand neomycin resistant gene (the plasmid was made by inserting SV40Large T antigen gene into a commercially available plasmid pGEM3SR αneo). Transfection was carried out by cultivating the cells for 1-3hours at 37° C. in a medium to which a mixture of plasmid andTRANSFECTAM (AR BROWN; cationic liposome) was added.

[0069] Following the transfection, cells were cultured for 2-3 days,allowed to detach by Trypsin-EDTA treatment and subcultured in 48-wellplate, when additional feeder layers (about 1×10⁴ cells/well) wereadded. After 1- to 2-day-subcultivation, the cells were re-transfectedwith the same plasmid. Then, the above procedure was repeated as theculture scale was stepwise increased to give immortalized cells.Afterward, the cultivation of resultant immortalized cells was performedusing 10% fetal calf serum-containing K1 medium in culture vessel coatedwith 0.1% gelatin without using feeder cells.

[0070] (4) Introduction of L-FABP Reporter Plasmid

[0071] The immortalized cells obtained in (3) above were culturedovernight. The cells were then co-transfected with the human L-FABPreporter plasmid (18 μg) prepared in Example 1 above and ahygromycin-resistant gene-containing plasmid (1.8 μg) (pPUR, Clontech,catalogue#6156-1). The transfection was carried out with TRANSFECTAM inthe same manner as descried in (3) above.

[0072] After transfection, the cells were cultured in a medium (10%fetal calf serum-containing K1 medium) supplied with hygromycin (100μg/ml). After 6-day-cultivation, the cells were passaged once andcultured for additional 10 days. The resultant hygromycin-resistantcolonies were then picked up under microscope. The picked up colonieswere seeded into 96-well plate and cultured, whereby several kinds oftransformed cell lines (No. 10-No. 19) containing the plasmid introducedwere obtained.

[0073] These cell strains were then cultured in high glucose-containingDulbecco's Modified Eagle's Medium (hereafter, “DMEM”) (Gibco)supplemented with 10% fetal calf serum.

[0074] (5) Morphological and Physiological Analyses of the Cell Line

[0075] The cell line obtained in (4) was subjected to analysis toconfirm whether or not they have morphological and physiologicalfeatures of proximal renal tubular epithelial cells, i.e., analysisregarding ability to respond to hormone and to uptake BSA.

[0076] Morphological observations of cell lines under a phase contrastmicroscope revealed that every cell line have pavement configurationcharacteristic to epithelial cells. Electron microscopic observation ofcell line No.13 revealed that the cell has microvilli at apical side,forms tight junction characteristic to epithelial cells, expresses actinand form hemidesmosome at basolateral side, and further, possesses cellpolarity in part. On the basis of these observations, the resulted celllines were identified as epithelial cells. The electron micrographs ofcell line No. 13 are shown in FIG. 1.

[0077] It is known that epithelial cells of renal tubule respondspecifically to a given hormone at respective segments and induceintracellular CAMP production. For example, proximal renal tubular cellsrespond only to parathormone (PTH) and not to vasopressin (AVP). On theother hand, collecting tubule cells, Henle's loop cells and the likerespond to AVP.

[0078] Each cell line (cell line No. 10, 13, 14, 15, 16 and 19) was thentested as to the reactivity to PTH and AVP according to the methoddescribed in reference example 1 (6) below. As a result, as shown inFIG. 2, every cell lines except for cell line No.10 responded to PTHstimulation and, especially, cell line No. 13 showed very highreactivity. On the other hand, no cell line responded to AVP. From theseresults, it was recognized that every cell lines except for cell lineNo. 10 possess characteristic features of proximal renal tubular cellsregarding reactivity to hormone and that cell line No. 13 especiallymaintains the features well.

[0079] A known proximal renal tubular epithelial cell line LLC-PK1 (ATCCCRL1392) was also tested as to reactivity to hormone in the same way asdescribed above. As a result, the ratio of cAMP amounts produced due tohormone stimulation were 1:0.7:7.4 for control:PTH stimulation:AVPstimulation, which showed that said cell line responds to AVP but not toPTH. That is, LLC-PK1, in contrast with cell line No. 13, does not havethe feature of proximal renal tubular cells regarding responsiveness tohormone.

[0080] The cell lines No. 13 and No. 16 were then examined as foralbumin uptake activity according to the method described in referenceexample 1 (5) below. It is known that proximal renal tubule is a segmentresponsible for reabsorption of protein in filtrate of glomerularfiltration and that proximal renal tubular cells are capable ofreabsorbing albumin and the like present in plasma in abundance. Asshown in FIG. 3, it was recognized that both cell lines have albuminuptake activity. Especially, cell line No. 13 showed about 5-foldenhanced albumin uptake as compared with control, indicating that cellline No. 13 maintains the feature of proximal renal tubular cell such asalbumin uptake and the like.

[0081] (6) Confirmation of Integrated Region of L-FABP Gene

[0082] As described above, cell line No. 13 is a mouse proximal renaltubular epithelial cell line to which an L-FABP reporter plasmid hasbeen introduced. A Southern blot analysis was performed according to thefollowing way to identify whether or not the reporter construct isintegrated into chromosome.

[0083] If the transcriptional regulatory region of human L-FABP genefrom reporter plasmid is inserted without deletion, the chromosomal DNAshould give 4.3 Kb fragment upon digestion with restriction enzymesApa-KpnI. Chromosomal DNA was prepared from cells of cell line No. 13,treated with the restriction enzymes Apa and KpnI and then applied to anagarose electrophoresis. DNAs were transferred to a nylon membrane(Hybond N+membrane, Amersham) from the electrophoresed gel, and themembrane was washed with 2×SSC and dried. Following prehybridization,the membrane was hybridized at 65° C. overnight in hybridization buffercontaining probes, washed with 2 ×SSC (containing 0.1% SDS) (65° C., 30min) and then subjected to autoradiography. A fragment (1.2 Kb)including 5′ upstream sequence of human L-FABP gene was RI labeled andused as a probe.

[0084] Consequently, 4.3 Kb fragment was detected and it was confirmedthat the transcriptional regulatory region of human L-FABP gene has beenintegrated without deletion. It was considered that cell line No.13contained L-FABP reporter plasmid-derived DNA construct (comprising thehuman L-FABP gene transcriptional regulatory region and luciferase gene)integrated into chromosome.

[0085] This cell line No.13 (hereafter, referred as “mProx37-13”) hasbeen deposited at The National Institute of Bioscience and HumanTechnology (1-1-3, Higashi, Tsukuba, Ibaraki, JAPAN) with the cell linename “mouse cell line mProx37-13” (Accession No. FERM BP-7038, transferdate to International deposition: Feb. 21, 2000).

Example 3 Assay of L-FABP Up-Regulating Activity in Proximal RenalTubular Epithelial Cells

[0086] A test substance was examined for the L-FABP up-regulatingactivity by reporter assay method in the following manner using theproximal renal tubular epithelial cell line mProx 37-13 (also referredas “cell line No.13”) comprising L-FABP reporter DNA construct and wasprepared in Example 2 above.

[0087] Firstly, the cell line mProx 37-13 was added in 96-wellflat-bottom plates at 5×10⁴ cells/100 μl/well and cultured. Thecultivation was conducted using plates pre-coated with 0.1% gelatin and,as a medium, a high glucose-containing Dulbecco's MEM medium (Dulbecco'sMEM, high glucose; DMEM) (Gibco) supplemented with 10% fetal calf serum,100 unit/ml of penicillin and 100 μg/ml of streptomycin. After48-hour-cultivation, the plate was washed once with serum-free medium,and a serum-free medium containing test substance was added thereto. Forcontrol samples, no test substance was added. After additionalcultivation, Luciferase activity was detected after 2 to 10 hours.

[0088] Luciferase activity was measured in the following way. Cells werewashed with phosphate buffered saline (PBS), added 20 μl of solution forcell lysis (LCβ-51, TOYO INK), allowed to stand for 20 minutes at roomtemperature and then stored at −80° C. overnight. After the plate storedat −80° C. was allowed to become room temperature, 100 μl ofluminescence substrate solution (PicaGene luminescence kit, TOYO INK)was added to initiate reaction. Amount of luminescence was detectedusing luminometer (MicroLumat LP98P, BERTHOLD). The luciferase activitywas obtained by integrating the relative luciferase units (RLU) for 10seconds from the beginning of reaction. The luciferase induction rate(Luc induction rate) was calculated based on the activity valueaccording to the following equation:

Luc induction rate=(test value)/(control value).

[0089] Induction of luciferase activity was observed when MCC-555,peroxisome proliferator-activated receptor (PPAR) agonist, or Etomoxir,carnitine palmitoyltransferase (CPT) inhibitor, was added as a testsubstance. These compounds were identified to have L-FABP up-regulatingactivity. The results of measurement are shown in FIG. 4.

[0090] MCC-555 (chemical name:5-[6-(2-fluorobenzyloxy)naphthalen-2-yl-methyl]thiazolidine-2,4-dione;EP604983) and Etomoxir (chemical name: ethyl2-[6-(4-chlorophenoxy)hexyl]oxiranecarboxylate; EP46590 and CurrentPharmaceutical Design, vol. 4, pp. 1-15, 1998) used herein weresynthesized according to the method disclosed in the literatures.

Example 4 Assay of L-FABP Up-Regulating Activity on Proximal RenalTubular Epithelial Cells

[0091] (Measurements at mRNA Level and Protein Level)

[0092] The plasmid isolated in Example 1, (2) above, which containshuman L-FABP chromosomal gene (entire length), was linealized andtransfected into the porcine renal tubular cell line LLC-PK1 or mouseimmortalized renal tubular epithelial cells prepared in a manner similarto that described in Example 2 (3) above to obtain stable transformedcell lines. The human L-FABP chromosomal gene was integrated into thesecells on their chromosome without deletion.

[0093] Using these cells, L-FABP up-regulating activity of testcompounds were examined at RNA level and protein level in the followingmanner. Cells were cultured to become confluent, when the medium wasreplaced with serum-free DMEM medium, and test compounds were added tothe medium at final concentration of 1 μM. The cultivation was continuedat 37° C. Control sample was obtained without adding any test compounds.

[0094] Measurement at mRNA level was carried out in the followingmanner. After cultivation, cells were collected and total RNA wasprepared. The resulted total RNA was used as a template for RT-PCR(Reverse transcriptase-Polymerase chain reaction) to detect human L-FABPmRNA. Also, in order to normalize the measurement values, GAPDH(glyceraldehyde 3-phosphate dehydrogenase) mRNA was detected as controlin the similar RT-PCR. The resulted PCR products were subjected toagarose-gel electrophoresis, the gel was stained with ethidium bromideto detect bands respectively and the expression amount was determined bydensitometry, or visually. As a result, when MCC-555 was added as a testcompound, induction of L-FABP mRNA was observed from 15 minute to 24hour after beginning of cultivation.

[0095] Measurement at protein level was carried out in the followingmanner. Cultivated cells were collected and sonicated and then cellextract was prepared. The amount of L-FABP in the extract was detectedby Western Blotting method or ELISA method with anti-human L-FABPantibody. As a result, when MCC-555 was added as a test compound, theup-regulating effect on L-FABP protein expression was observed. Theup-regulating effect on L-FABP protein expression was also observed whenEtomoxir was added.

Example 5 Assay of L-FABP Up-Regulating Activity on Human UrinaryExfoliated Cells

[0096] (Assay at mRNA Level)

[0097] (1) Isolation of Human Urinary Exfoliated Cell

[0098] Renal tubular epithelial cells exfoliated from kidney tissuesinto urine (hereafter, referred to as “urinary exfoliated cell”) wereisolated from human urine sample and cultured in the following manner.

[0099] Firstly, urine samples from male infant nephritis patients (0-3years old) were collected using the clean-catch method. The urinesamples were then centrifuged (1000 rpm, 10 minutes) at room temperatureand the supernatant was discarded. The pellet was washed with 10% (v/v)fetal calf serum-containing Dulbecco's modified Eagle's medium twice andthen with culture medium once. The culture medium used herein was amixture (1:1) of Dulbecco's modified Eagle's medium and Ham's F-12medium, to which mixture was added fetal calf serum (10% v/v), insulin(5 μg/ml), transferrin (5 μg/ml), sodium selenite (5 ng/ml),dexamethasone (10⁻⁸ M), nicotinamide (5 mM), penicillin (100 IU/ml) andstreptomycin (100 μg/ml).

[0100] The pellet was suspended into the culture medium, dilutedappropriately and then cultured in collagen type 4-coated dish (3.5 cmin diameter, Falcon). The cultivation was performed under the conditionof 5% CO₂, 37° C.

[0101] The cultured cells were confirmed to be renal tubular epithelialcells because domed form was observed as the cells became confluent and,further, the cells were positive to alkaline phosphatase staining.

[0102] (2) Assay of L-FABP Up-Regulating Activity

[0103] A test compound was examined for the L-FABP up-regulatingactivity at mRNA revel in a manner similar to that described in Example4, as shown below.

[0104] Firstly, cells were cultured confluently and the medium wasreplaced with Dulbecco's Modified Eagle's Medium (serum-free) suppliedwith a test compound at final concentration of 1 μM. Cultivation wascontinued at 37° C. Test compounds used were MCC-555 (PPAR agonist) and4-THA (2-hydroxy-3-propyl-4-[6-(tetrazol-5-yl)hexyloxy]-acetophenone)(CPT inhibitor). For control sample, no test compound was added.

[0105] After cultivation, cells were collected and total RNA wasprepared. The resulted total RNA was used as a template to performRT-PCR (Reverse transcriptase-Polymerase chain reaction). The resultedPCR products were applied to agarose-gel electrophoresis, the gel wasstained with ethidium bromide to detect bands respectively and theexpression amount was identified by densitometry or visually. Also, inorder to normalize the measurement values, rBAT (related to b^(o,+)system amino acid transporter; Purroy et. al., Genomics, Vol 37. pp.249-252, 1996) mRNA was detected as control by RT-PCR.

[0106] Consequently, as shown in Table 1 below, the amount of L-FABPmRNA expression in human urinary exfoliated cells (human renal tubularepithelial cells) was increased when MCC-555 or THA was added. On thebasis of these results, these compounds were identified to have L-FABPup-regulating activity also in human proximal renal tubular cells. TABLE1 Test compound L-FABP mRNA rBAT mRNA None − + MCC-555 + + (1 μM) 4-THA(10 μM) + +

Example 6 Effect of Suppression of L-FABP Expression on Cytotoxicity

[0107] The effect of suppression of L-FABP expression on cytotoxicitywas examined with hypoxia/reoxygenation model (in vitro renalischemia/reperfusion model) using porcine proximal renal tubule-derivedLLC-PK1 cells in the following manner.

[0108] Firstly, porcine L-FABP cDNA was cloned by RT-PCR, subcloned inpRC/CMV to obtain antisense RNA expression vector pLFABP-pRC/CMV.

[0109] Then, LLC-PK1 cells were seeded in 6-well plate (1×10⁵cells/well), cultured for 24 hours and transfected with pLFABP-pRC/CMVusing Lipofectamine (GIBCO). As control, cells were transfected with thevector (pRC/CMV) only. These cells were cultured for 24 hours,transferred to an anaerobic chamber (COY) and exposed to hypoxia for 24hours. Cells were returned to normal atmosphere and reoxygenated for 24hours, and the cytotoxicity was then measured.

[0110] The cytotoxicity was measured utilizing LDH (lacticdehydrogenase) leaked into medium as an index. That is, after hypoxicexposure, the medium was collected and the LDH activity contained in themedium was measured using the assay kit “LDH-Cytotoxic Test Wako” (WakoPure Chemical Industries, Ltd.)

[0111] As a result, as shown in FIG. 5, the leaked LDH activity afterhypoxia/reoxygenation was higher in the cells transiently expressingL-FABP antisense RNA than control cells transiently transfected withvector alone, indicating that the former cells suffered more seriousinjuries.

[0112] The fact that the expression of L-FABP antisense RNA, i.e.suppression of L-FABP expression, increased the cytotoxicity duringhypoxia/reoxygenation led to a view that L-FABP has a function ofprotecting cells under a condition that injures proximal renal tubularcells, such as renal ischemia/reperfusion and the like.

Example 7 Effect of Imposed Expression of L-FABP on Cytotoxicity

[0113] The effect of imposed expression of L-FABP on cytotoxicity wasexamined with hypoxia/reoxygenation model (in vitro renalischemia/reperfusion model) using proximal renal tubular cells in amanner similar to that described in Example 6 above.

[0114] The plasmid comprising human L-FABP chromosomal gene (entirelength) isolated in Example 1, (2) above was linearized and transfectedinto the mouse proximal renal tubular cells (clone 24) obtained by thesame manner as Example 2 (3) to obtain a stable transformed cell (clone24-19). The human L-FABP chromosomal gene was integrated into the cellon the chromosome without deletion.

[0115] The clone 24-19 obtained above and clone 24 as control cell wererespectively exposed to hypoxia in an anaerobic chamber (COY) for 24hours. Cells were returned to normal atmosphere and reoxygenated for 24hours, and then the cytotoxicity was measured. The cytotoxicity wasmeasured utilizing LDH (lactic dehydrogenase) leaked into medium as anindex.

[0116] As a result, as shown in FIG. 6, the leaked LDH activity afterhypoxia/reoxygenation is lower in cells (clone 24-19) introduced withhuman L-FABP gene to be expressed than in control cells (clone 24),indicating that former cells are more resistant tohypoxia/reoxygenation. The fact that the imposed expression of L-FABPincreased the resistance to cytotoxicity during hypoxia/reoxygenationled to a view that L-FABP has a function of protecting cells under acondition that injures proximal renal tubular cells, such as renalischemia/reperfusion and the like.

Example 8 Effects of Drug on Model Mouse of Renal Diseases

[0117] An adriamycin-induced glomerular sclerosis mouse was used as arenal disease model to confirm effect of PPAR agonist MCC-555 in thefollowing manner.

[0118] An adriamycin(AD)-induced glomerular sclerosis mouse is apathological model which develops focal glomerular sclerosis in 3 weeksafter the onset of nephrosis symptom while showing the symptom andeventually results in irreversible chronic renal insufficiency (Chen et.al., Nephron, vol. 78, pp.440-452, 1998). As shown in Examples 3, 4 and5, MCC-555 is a compound confirmed to have L-FABP up-regulatingactivity.

[0119] In the experiment, 7-week-age female BALB/c mice (purchased fromCharles River Japan, Inc.) were used. At the first day of experiment(Day 0), Adriamycin (Sigma) was administered intravenously at 10 mg/kg(10 ml/kg dissolved in saline at 1 mg/ml). Normal group receivedintravenously an equivalent amount of saline only. To the mice wereadministered orally MCC-555 or carrier alone for 14 days from the firstday of experiment (Day 0 to Day 13). The administration was initiallyconducted 1 hour before the adriamycin administration, andadministration volume was 10 ml/kg for each time.

[0120] In drug administration group (n=6), MCC-555 (suspension in 0.1%Tween 80-containing purified water) was administered at a dose of 10mg/kg to mice that have received adriamycin. In control group (n=6),carrier (0.1% Tween 80-containing purified water) alone was administeredto mice that have received adriamycin. Also in normal group (n=3), micereceived carrier alone.

[0121] Mice were separately housed in “a metabolic cage” and allowed toaccess food and water ad libitum while collecting urine for 24 hours.Specifically, collection of urine was performed 4 times in total, i.e.,day 3-4, day 6-7, day 9-10, and day 13-14. Following measurement ofvolume, the collected urine was subjected to determination of urinaryprotein, NAG (N-acetyl-β-D-glucosaminidase) and creatinine (CRE)concentrations by clinical chemistry analyzer (SuperZ-818, NITTEC Co.)After the collection of urine on the last experiment day (Day 14), bloodwas collected via abdominal aorta under ethereal anesthetization andserum was separated. Cholesterol and albumin were determined usingAutomated clinical analyzer TBA-80FR (TOSHIBA). Also, kidneys wereisolated and fixed with neutral buffered formalin.

[0122] The results of experiment are provided in FIG. 7. As shown inFIG. 7, in control group, urinary protein and NAG excretion amount thatis an index of proximal renal tubular injury were increased at 7 to 14days after AD administration. In contrast with this, in drugadministration group, the increase of urinary protein and NAG excretionamount were significantly suppressed. The results of blood biochemicalexamination at Day 14 are provided in FIG. 8. As shown in FIG. 8,hypercholesterolemia and hypoalbuminemia were recognized in controlgroup, which are main symptoms of nephrosis. In contrast with this, suchsymptoms were improved in drug-administration group.

[0123] As described in above, MCC-555 suppressed NAG excretion that isan index of proximal renal tubular injury, and also improvedproteinuria, which is main symptom of nephrosis, and blood biochemicalexamination parameters in renal disease model mouse (adriamycin-inducedglomerular sclerosis model). Furthermore, it was confirmed that MCC-555also inhibited histological changes of kidney in the same model mouse bytissue imaging examination.

Example 9 Effects of Drug on Renal Disease Model of Transgenic MouseIntroduced with Human L-FABP Gene

[0124] Transgenic mouse (hFABP-Tg mouse) introduced with human L-FABPgene was prepared. The hFABP-Tg mouse was used to make renal diseasemodel similar to that of Example 8 and the effect of MCC-555 wasidentified.

[0125] In the preparation of hFABP-Tg mouse, male BCF1 mouse (13-week orover) was used for sterile mating and natural mating; female ICR mouse(10-week or over) for embryo transplant and as a foster mother; maleBDF1 mouse (13-week or over) for mating; and female BCF1 mouse (8-weekor over) for collection of eggs, respectively. The resultant transgenicmouse (B6C3F1 line) was used to backcross with BALB/c mouse.

[0126] The resultant female transgenic mouse was used to prepare a renaldisease model (adriamycin-induced glomerular sclerosis model) and toconfirm effects of drug (MCC-555). The preparation of disease model andconfirmation of effect of a drug were conducted in a similar manner tothat described in Example 8 above except that the dose of adriamycin onthe first experiment day (Day 0) was 15 mg/kg (15 ml/kg, dissolved insaline at concentration of 1 mg/ml).

[0127] In drug administration group (n=5), MCC-555 (10 mg/kg) wasadministered to mice that have received adriamycin, and in control group(at the beginning of experiment, n=5), only carrier was administered tomice that have received adriamycin, In normal group (n=3), mice thatwere not treated with adriamycin received carrier. Collection of urinewas performed 3 times in total, i.e., day 6-7 day (Day 7), day 10-11(Day 11), and day 13-14 (Day 14) and urinary protein, NAG and creatinineconcentrations were determined by Automated clinical analyzer aftermeasurements of urine volume.

[0128] The results of experiment are provided in FIG. 9. As shown inFIG. 9, in the control group, urinary protein and NAG excretion amountwere increased after AD administration, whereas, in drug (MCC-555)administration group, such increase was significantly suppressed.Further, in the control group, 3 animals died during the experiment butin drug (MCC-555) administration group, no animals died.

Reference Example 1 Isolation of Proximal Renal Tubular Epithelial Cellsby Density Gradient Centrifugation Method and Primary Culture Thereof

[0129] (1) Isolation of Proximal Renal Tubular Cells

[0130] Renal cortical cells were isolated from mouse kidneys in thefollowing manner according to density gradient centrifugation methoddescribed in Vinay et. al., American Journal of Physiology, vol. 241,F403-F411, 1981.

[0131] Two mice were decapitated and bled, and the kidneys were isolatedand washed with cold Hank's buffered solution (HBS) and then capsula wasremoved. Only the cortex was then excised from capsula and cut into 1-2mm cubic. The sectioned tissues were washed with HBS briefly, and 5 mlof collagenase type 4 solution in HBS (400 μg/ml) was added. The mixturewas incubated at 37° C. for 30 minutes. After stirring for 5 minutes,the mixture was filtrated thorough nylon mesh (100 μm) to remove tissueaggregates and washed with HBS. One ml of HBS was added and pipettedbriefly to prepare the cell suspension.

[0132] The resultant cell suspension was layered on 30-50% Percollgradient solution and centrifuged (3,500 rpm, 4° C., 30 minutes). Aftercentrifugation, cells separated into each layer (4 layers from the top,fractions 1 to 4) of Percoll density gradient were recovered carefully.

[0133] (2) Alkaline Phosphatase Staining of Separated Cells

[0134] In the case of kidney, alkaline phosphatase is known to belocated in proximal renal tubule. Accordingly, a portion of cells ineach layer recovered in (1) above were harvested and stained withalkaline phosphatase as described below. That is, the cell suspensionwas centrifuged, immobilized on preparation, stained with alkalinephosphatase substrate kit (FUNAKOSHI) and then embedded into glycerol.As a result, the uppermost layer (fraction 1) showed the highestalkaline phosphatase positive rate and hence was considered to behighest in the content of proximal renal tubular cells.

[0135] (3) Primary Culture of Proximal Renal Tubular Epithelial Cells

[0136] The cells of fraction 1 described in (1) above were treated withcollagenase as described below and then primary-cultured. That is, thecollected cells were washed once with HBS and treated with enzyme byadding 1 ml of a collagenase type 4 solution (2 mg/ml in HBS) andincubating at 37° C. for 5 minutes. After washing twice with HBS,incubation was continued.

[0137] It is generally known that the primary culture of epithelialcells is contaminated with fibroblasts. Accordingly, it is necessary toinhibit the growth of fibroblasts while propagating epithelial cellsselectively. To this end, for the cultivation, a combination of (i)MEM/D-Val medium (wherein L-valine was replaced with D-valine in MEMmedium; Gibco) supplemented with fetal calf serum, which selectivelyinhibit the growth of epithelial cells and (ii) serum-free K1 medium,which contains hormones and growth factors capable of specificallypropagating epithelial cells, but lacks factors participating in thegrowth of fibroblasts (50:50 DMEM/Ham's F-12, 15 mM Hepes, 13.4 mMsodium bicarbonate, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/mlselenous acid, 0.05 μM hydrocortisone, 10 ng/ml epidermal growth factor)was used in the following manner.

[0138] First, the cells were cultured in MEM/D-Val medium containing 10%fetal calf serum for 1 to 2 days. After the confirmation of adhesion andgrowth of cells, the medium was replaced with serum-fee K1 medium, andthe cultivation was continued for about 1 week. The cells were seededinto plates pre-coated with 0.1% gelatin and cultured within CO₂incubator.

[0139] (4) Morphological Analysis of Cells

[0140] When the cultured cells described in (3) above were observedunder a phase contrast microscope, cell growth with pavementconfiguration was recognized. Further, when the cells became almostconfluent, the domed forms were recognized. These observations suggestthat the intra-epithelial water and solute transport being performed.From these facts, it was confirmed morphologically that the resultedcultured cells were renal tubular epithelial cells.

[0141] (5) Albumin Uptake Activity

[0142] It has been known that the proximal renal tubular cells havealbumin uptake activity. The cultured cells described in (3) above wereexamined for albumin uptake activity in the following manner. Cells werecultured to become confluent in 10 cm dish, when the medium was replacedwith MEM/D-Val medium and the cells were cultured overnight. Bovineserum albumin (BSA) was then added to the medium at the finalconcentration of 50 mg/10 ml and the cultivation was continued at 37° C.for 90 minutes. As control, cells were incubated on ice in the samemedium containing BSA. After cultivation, the cells were washed withphosphate buffered saline (PBS) (pH 7.4) and recovered. These cells weresuspended into 200 μl of PBS (pH 9.6) containing protease inhibitors andsonicated. To the sonicated solution was added PBS (pH 6.4) to adjust pHwithin the neutral range, and the solution was centrifuged at 12,000 rpmfor 15 minutes and the supernatants were collected as cell extracts. Theresulted cell extracts were assayed for the presence of BSA by ELISA.

[0143] In the ELISA method, anti-albumin antibody (rabbit polyclonalantibody, Sigma) was used as the primary antibody and biotin-labeledanti-albumin antibody was used as the secondary antibody. The primaryantibody was absorbed onto 96-well immunoplates. After addition of thecell extracts (100 μl/well), incubation was conducted at roomtemperature for two hours. To the well was added diluted secondaryantibody and incubated at room temperature for two hours. After washingthe wells, color detection was conducted with a detection kit (Vectorlaboratories, Vector Stain ABC-PO kit) containing streptavidin andbiotin-labeled horseradish peroxidase.

[0144] As a result, the BSA uptake was recognized to be 2.3-fold whencultivation was conducted at 37° C. with addition of albumin comparedwith control. It was confirmed that the cells have physiologicalfunctions of proximal renal tubular cells.

[0145] (6) Responding Activity to Hormone

[0146] The proximal renal tubular cells are known to be responsive toparathormone (PTH) and induced to produce intracellular CAMP butunresponsive to vasopressin (AVP). The cultured cells described in (3)above were used in the examination of responsiveness to hormone in thefollowing manner.

[0147] The cells were cultured to confluent in 24-well plate, when thecell surface was washed with a medium (MEM/D-Val medium) twice. To thecell was added a medium supplied with either PTH (10⁻⁷ M) or AVP (1U/ml), and IBMX (3-isobutyl 1-methylxanthin; cAMP phosphodiesteraseinhibitor) (10⁻⁴ M). For control, a medium supplied with IBMX alone wasadded. After incubation at 37° C. for 10 minutes, CAMP in cells and inmedium was extracted with 65% ethanol. The amount of CAMP was determinedby ELISA (CAMP EIA system kit, Amersham).

[0148] As a result, approximately 1.6-fold intracellular CAMP wasrecognized in the case of PTH stimulation compared to control. From thisobservation, it was confirmed that proximal renal tubular cells werecontained in the primary culture. Further, in the case of AVPstimulation, approximately 4.6-fold CAMP was recognized compared tocontrol, which indicated that cells originated from sources other thanproximal renal tubular cells might be blended. SEQUENCE LISTING FREETEXT Free text of SEQ ID NO: 1 <223> GATA signal (GATA_signal) <223>peroxisome proliferator responsive element (PPRE) <223> hepatic nuclearfactor (HNF) binding-site <223> hypoxia inducible factor (HIF)binding-site

INDUSTRIAL APPLICABILITY

[0149] According to the method of the present invention, it is possibleto screen novel therapeutic and/or prophylactic agents for renaldiseases precisely and efficiently. Further, such therapeutic and/orprophylactic agents identified and characterized according to thepresent method (i.e., therapeutic and/or prophylactic agents of thepresent invention) are useful as excellent therapeutic and/orprophylactic agents for renal diseases of which mechanism of action isdefinite. In addition, the novel cell line of the present inventionmaintains well the physiological properties as proximal renal tubularepithelial cells and are useful in various aspects such as research anddevelopment of therapeutics for treating renal diseases or the like.

1 1 1 5357 DNA Homo sapiens CDS (4767)...(4835) intron (4836)...TATA_signal (4691)...(4697) misc_signal (4136)...(4141) GATA_signal 1gtcgactgca gtcaacggat cctctccrty ctctvaccct caccctcaac tacgccccag 60tgtctgttgt tcccctacta gtatccatgt gttctcattg tttagctccc acttataagt 120gagaacacac agtatttaat tttctgttcc tgtgttcgtt tgcttaggat aatggcctct 180agctccatcc acgttgctgc aaaggacatg atctcatgct tttttatggc tgcatcatat 240tccatggtgt atgtatacca cgctttcttt attcagtcta ctgttggtgg gcattaggtt 300gattccatgt ctttgctatt gtgaataatg ctgcaatgaa catactcatg catgtgtcct 360tatggtagaa caatttatat tcttttgggc atatacccag tagtgggact gctgggtcaa 420atggtaattc tgttttaagt tctttgagga atcaccacac tgctttccac aatggctgaa 480ttaattcaca ctctcaccag tagtctgaaa tgttcctttt ctctgcaacc ttgccagcat 540gttatttttt gacttttaat agtagccatt ctgactggtg tgtgatagta tctcattgtg 600gttgtgattc gtttacgcaa ttatttcaca aggcaaaaaa tgttgtgacc atttaacaga 660taagcaagta gaggctcacc aaggctccac atcaggtata aagaagagct gggcccaaac 720ccaagtcctt tccctctgca gccctgcctg tagcagaatt atgggctcct cgggccacct 780cctatgttca aaacacccag gaaatgggca ggctggcatt gcaggcactg aggtcaaagg 840cgcttgttct aaaatgaaca cctgtcatat cctggccacc aagaggtgac tcccctcctt 900tcccacacct gcacgctgcc cgtctgagta gggatgtgag aagaggggat gtctagtgtg 960gagtgagcag ggccagaatc tttggacact ccaaagctgt gcccacagag tgggagtctc 1020tttttttctt tcttgaaagc cacttgaact ttgtcattgc aattaagctt atctcttctt 1080gtgtttgctc agctagtggg tggaagatgt cattaccctc tcctaacttt atggaaaact 1140catgagtatt agtttttgaa atggctactg atctctgtgc cctcaagatt cacacacaca 1200caaaaaaaaa gacaaaggga agtcatcggg ggttgcctaa gaatggccat atctcagacc 1260tgggggagag ggggagtcct ggcctggcgt tgatcagctc aatttacttg tcaggctgcc 1320catctgccag ggaaactgtc aaggcttcac tggttgtgcc aggcccttga ccaacctctg 1380ggtcagctgc attcccaatg ctgttctgag tctgcagcgt tgcctggaca gggtaggcag 1440gaagtgcagg gtgaggcaga gtggtccaca aggagacagc tcatgggtcc tgctgttcct 1500cagacaccag gaaaggtgca cctggtccct acttcacctg aggcctccgg ggttggtgct 1560gacttgctct atggagaagc tttcatgacg ctccttccca ctcccctccg tagatcccag 1620aaagcagcag tgtggggcat gatacttgac atttgcgatc aaggggagag ggcctagttg 1680tggaatgtat aagccagaca tccctttatc tgagcgggat gtcaggcagg cagctggggg 1740catgagataa catgggcact tggagcagat tctcccaaac gaagcaactg atgggtgtga 1800tgggagttgg gcatggtaat gactctatca cggtcatttt cctcggtgat tatttcattg 1860attttaggct gcaaaactcc taagctcccc atctaagcaa gtgtttacgt attccttcct 1920tgacaaatgc tagaagagct ctttaggtag gtagtgaata ggatctcagg tatagagctg 1980aatttagccc tgggctgaac cttcacatgg tcattagaca gacatatgcc agctattggt 2040gcaggcttta ggtgtcacat gtaacccaga aggatctaaa taaaatgacc ttgaattcct 2100ccaaagccct tcctaggatg tctagggacc tggggaaccc tgtagggaat gcatccccac 2160agacactcca cttcctgcag tctgggggag ggacaggaag acacacccag cctctcctgg 2220tctcctgcat gagggagctg cccttctctt ggtactgctt ctgcatgtgc catctcagcc 2280tcatcacatc catccagaaa taaagctcag ccttgcatca ttttccactt gctcacatat 2340tgattcaggc actatatttt actgaagctt ttgaaatagt tggggtggaa attaaagaaa 2400gatggatggg gattcactgc agcaaatgag cagcttcaaa gccaatgcca tcctggagct 2460cctttctgca gtctggggga gggacaggaa gacacaccca gcctctcctg gtctcctgca 2520tgagggagct gcccttctct tcaccagctt ggctctcgct gacacacggg tctgaggtct 2580ggggtctcac acactctgtc tcccaggcag ctaatccagt catttacaca cacacacaca 2640cacacacaca cacacacaca cactaggaat tctcatgcca gttcttaaga caatggattt 2700tttacttcct tgcatataca aaaatgaaaa atcagcatcc catctggaga ccagggtcta 2760atatggaccc ctttagtaca atagacatgt taaaagatgt ataatcaaag ctcagagcaa 2820gatgagagaa ggactgtggg tgtgcccata cttctttgag ccacctcgcc ccttcctgcc 2880cgctgttcag gtagtcgtgg tgattataaa aagccataag ttctcagcat gcaggcccac 2940ttcgcttgct gcagggacag ccccaattca tggcctggga ggcaccttgt gtcctagaca 3000ctgtcagagg agggccaagc agggagggcc agagcacccc tctgtgctgg agggaaaggt 3060ctgctggtgc cagaatctgc atggcaggac actccattag tgagtgttct ctgcccacag 3120tgctccaaag gtgaggacct ccctgtggca gggtacacct taacaccacc tgtcagggat 3180gctgtagaag gaccctggca ctgggactgt gggtggagga gagaacttct gaagtgcttt 3240gctggatcaa catgtccaaa tgctggagac cacggagggc agagagagtg gcatggacgc 3300ccccgatgtg tccacttggc cctgtggctg gaactgtggc ttgaggagaa ggaaggaaaa 3360aggatggaaa ggtagactgg agttgccttt tgagggctgg cagccctgct agaagtttag 3420gcaatgggag tcattttcca agccaaaaat tagaactcat gatagggaaa tggaagtgtt 3480attgggggtg gccatcatga cccgtcacgt gacactaggc catccaggac ctgtggcctc 3540tgaagtgatg ggaaccactg aggctcatgc ctggagcagt ggcccgactc agcctatgct 3600tcaggatgat ctgtctaacc acagagtaga ggaggcccag aggatgaagg ctggaggctg 3660caggtgaaaa ggttgacgcc aaagtccaga caaagaaggg agagaatcta ggggtgaggg 3720ggtgcttgta aagagctgcc tcagaggcag gaactgggac gtgcacccat tgggcaccac 3780gcctctctgt gcttcacaac aaactggcac atcccaaggc cactggaagc cctgctgggc 3840catctcccca aggccagtgc tgtacacata accctacaag accagtttcc tacacataac 3900cctacaagac cagtttccta cacataaccc cagatccctt gtcctgtctt ctcagtgggg 3960ctggagcaag tcagcaggtg ccactttctc ctgccttgtc tctgcctaat aaaatgcgtc 4020tcaatgtttt acacctgcca tttagcatgg actgctttaa cacctcaaaa aggcctgtgg 4080aggagcctat aatcatcaag gaggaattcc cagaatacaa aataacacta gcggctgata 4140acaactctaa aaaataagtt tgtgtaataa tgggggtgag aagagatcat aaggttatgt 4200aaataaggtg aggttttgag ttcaaaggaa ttctctggta tttttctgtg tgtgtacaca 4260tgcacccaca cacttgtgtg tatatgtgta cagacatata taaacacatg catataatgt 4320gtatatatgc attacatata tgcacattca tacatcttta tgtacaaaat acatatatgt 4380atatataaac accgatgtac aaacacatac gcacacatct atatacatac acatgtgtgt 4440gcacatatac acatacctgc atatacacac atttcgtggg gtgcggagag tcacttaaag 4500gctgcagggc cataaggctt cctgcttgac tgatattcat taatgtttgc tgaattacag 4560caaacctttg ctgtgcccat cctgttcttt atcattgacc attgctctca ggagttaatg 4620tttgaacctg gccataaagg aatcaacagc tgctgacctc tggccgctat tcgaagggaa 4680gggagccccc tataaaacag cctacagtgg acagtctggt cggcagagcc gcaggtcagt 4740cgtgaagagg gagctctatt ggatccatga gtttctccgg caagtaccaa ctgcagagcc 4800aggaaaactt tgaagccttc atgaaggcaa tcggtgagtg ctggactgaa aggcaaagct 4860gtgggtcaca tcagtgaggg tctagctcta ccagcagtgg ctatttaggg tccaaatgtt 4920gagatggggg gagagattca ggctatatac acaaactagg gggcatttta ctgacttctg 4980gagttatttg cagcaagtcc tcactgcaca aggccacccc ataggcaagt ggaaagagca 5040ctgaactagg gtgtggtctg gattgatggc catgtcattg gcacttcgtt gtgtgatcct 5100attcctgcat ttattcattc ttcaaacaaa cagtgattca aggccaacca cacactgagc 5160actgtgctgg atagatacca gggagacaga tgataaaggc aataaggcat tttcttatca 5220gggaagagaa gagaagaaat aaaggtaata aggcattccc tcaggtgtat ggagcgctga 5280ctgtgccagg cgactgtgct gaccatggga tatgcaatac cccatctatt ccttgccaaa 5340aaactaagct gggtacc 5357

1. A therapeutic or prophylactic method for renal disease not caused bydiabetes, which comprises administering to a patient in need thereof ofan agent comprising, as an active ingredient, a compound having activityof peroxisome proliferator-activated receptor (PPAR) agonist.
 2. Thetherapeutic or prophylactic method of claim 1, wherein the renal diseaseis selected from the group consisting of glomerulonephritis, nephroticsyndrome, focal glomerulosclerosis, immune complex nephropathy, lupusnephritis, drug-induced renal injury and renal insufficiency.
 3. Thetherapeutic or prophylactic method of claim 1, wherein the renal diseaseis selected from the group consisting of nephrotic syndrome, focalglomerulosclerosis and drug-induced renal injury.
 4. The therapeutic orprophylactic method of claim 1 wherein the compound having activity ofPPAR agonist is MCC-555.
 5. A method for screening or identifyingtherapeutic or prophylactic agents for renal disease, which comprisesthe following steps of: (A) assaying a test substance for the activityof up-regulating the expression of liver-type fatty acid-binding protein(L-FABP) in animal cell, wherein said assay comprises the step: (1)culturing animal cell in the presence or absence of the test substance;and comparing the amount of L-FABP expressed in the cell in the presenceof the test substance with the amount of L-FABP expressed in the cell inthe absence of the test substance; or (2) administering the testsubstance to an animal and comparing the amount of L-FABP expressed inrenal tissues or cells of the animal with the amount of L-FABP expressedin renal tissues or cells of a non-treated animal; and (B) selecting oridentifying a substance having L-FABP up-regulating activity as acandidate therapeutic or prophylactic agent for renal diseases.